Detecting target nucleic acid sequences is a technique that is being used to a greater and greater extent in many fields, and the range of applications of that technique is predicted to widen as it becomes more reliable, cheaper and faster. In the human health field, detecting certain nucleic acid sequences can in some cases provide a reliable and rapid diagnosis of viral or bacterial infections. Similarly, detecting certain genetic peculiarities can allow susceptibilities to certain diseases to be identified, or provide an early diagnosis of genetic or neoplastic diseases. The detection of target nucleic acid sequences is also used in the agroalimentary industry, in particular to provide product traceability, to detect the presence of genetically modified organisms and to identify them, or to carry out food checks.
Detection procedures based on nucleic acids almost systematically involve a molecular hybridisation reaction between a target nucleic acid sequence and one or more nucleic acid sequences complementary to that target sequence. Such processes have a number of variations, such as techniques known to the skilled person as “transfer techniques” (blot, dot blot, Southern blot, Restriction Fragment Length Polymorphism, etc.), or such as miniaturised systems on which the complementary sequences of the target sequences are previously fixed (microarrays). Within the context of such techniques, complementary nucleic acid sequences are generally termed probes. A further variation, which can in itself constitute the basis of a diagnostic procedure or may simply be a supplementary step in one of the techniques mentioned above (in particular to increase the concentration of the target sequence and thus, the sensitivity of the diagnosis), consists of amplifying the targeted nucleic acid sequence. A number of techniques that can specifically amplify a nucleic acid sequence have been described, the most popular technique being the Polymerase Chain Reaction (PCR). Within the context of that technique, complementary nucleic acid sequences of target sequences, termed primers, are used to amplify those target sequences.
PCR reactions involve repeated cycles, generally 20 to 50 in number, and each is composed of three successive phases, namely: denaturation, primer annealing, strand elongation. The first phase corresponds to transforming double-stranded nucleic acids into single-stranded nucleic acids; the second phase is molecular hybridisation between the target sequence and the complementary primers for said sequence, and the third phase corresponds to elongation of the complementary primers hybridised to the target sequence, using a DNA polymerase. Those phases are carried out at specific temperatures: generally, 95° C. for denaturation, 72° C. for elongation, and between 30° C. and 65° C. for annealing, depending on the melting temperature (Tm) of the primers used. It is also possible to carry out the annealing and elongation steps at the same temperature (generally 60° C.).
Thus, a PCR reaction consists of a sequence of repetitive thermal cycles during which the number of target DNA molecules acting as the template is theoretically doubled for each cycle. In practice, the PCR yield is less than 100%, so the quantity of product Xn obtained after n cycles is:Xn=Xn−1(1+rn), where
Xn−1 is the quantity of product obtained in the preceding cycle, and rn is the PCR yield in cycle n (0<rn≦1).
Assuming the yield to be a constant, i.e., identical for each cycle, the quantity of product Xn obtained after n cycles from an initial quantity X0 is:Xn=X0(1+r)n   (A)
In practice, the yield r reduces during the PCR reaction, due to a number of factors such as a limiting quantity of at least one of the reagents necessary for amplification, deactivation of the polymerase by its repeated passes at 95° C., or its inhibition by pyrophosphates produced by the reaction.
Because of this reduction in yield, the PCR reaction kinetics firstly exhibit an exponential phase (where r is a constant), which then changes into a plateau phase when r reduces.
During the exponential phase, equation (A) above applies, and can also be written as:log(Xn)=log(X0)+n log(1+r)
Thus, in the exponential phase of the PCR, the curve showing the quantity of product on a logarithmic scale as a function of the number of cycles is a straight line with slope (1+r) which intersects the ordinate at a value equal to the logarithm of the initial concentration.
Real-time measurement of the quantity of product obtained can thus provide the initial concentration of the template, which is of particular importance in a large number of applications, for example when measuring the viral charge in a patient, or to determine the variability of a transcriptome.
Generally, the PCR employs reaction volumes of 2 μl to 50 μl and is carried out in tubes, microtubes, capillaries or systems known in the art as “microplates” (integral assemblies of microtubes). Each batch of tubes or equivalent containers must thus be successively heated to the three temperatures, corresponding to the different phases of the PCR, for the desired number of cycles.
Using tubes or similar systems obliges the operator to carry out many manipulations to prepare as many tubes and solutions (known in the art as mix PCR) as there are target sequences to be amplified, even when using a single sample of nucleic acids, with the exception of multiplex amplification procedures, which amplify a plurality of target sequences simultaneously in the same container, either using low specificity primers that can hybridise with a plurality of target sequences, such as RAPD—random amplified polymorphism DNA, or using specific primers in larger numbers, where each pair of primers used amplifies a single target sequence. Multiplex amplifications correspond to particular cases and are not in routine use. Further, they do not guarantee freedom from interactions of one amplification reaction with another, and because of possible hybridisations between primers, can only be very limited in the number of target sequences amplified per container.
Those different manipulations cause a number of disadvantages.
Firstly, they are time consuming. Secondly, they are not risk-free as regards possible contamination from one tube to another or from the external environment (dust, bacteria, aerosols or other contaminants that may contain nucleic acid molecules or molecules that may influence the efficacy of the amplification reaction). Further, homogeneity of volume and reagent concentration from one tube to another is not guaranteed. Finally, the volumes are necessarily manipulated manually and are generally greater than 1 μl, which affects the costs of carrying out PCR as the reagents employed are expensive.
The use of devices designed for at least partial automation of such manipulations can overcome some of those disadvantages. However, those instruments are relatively expensive and their use is, therefore, only economically justified when carrying out many PCR amplifications, for example for genome sequencing.
Some instruments also exist that can carry out kinetic PCR amplifications. As seen above, kinetic PCR necessitates real-time, specific quantification of the amplified target sequence. The use of a fluorescent reporter in the reaction mixture allows the increase in the total quantity of double-stranded DNA to be measured in that mixture. However, that method cannot discriminate amplification of the target sequence from background noise or from possible non specific amplification. Several probe systems have recently been described that specifically measure amplification of a set target sequence. They are based on complementary oligonucleotides of that sequence, and bonded to pairs of fluorophore groups or fluorophore/quenchers, such that hybridisation of the probe to its target and the successive amplification cycles cause an increase or reduction in the total fluorescence of the mixture, depending on the case, proportional to the amplification of the target sequence.
Examples of probes that can be used to carry out kinetic PCR that can be cited are the TaqMan™ (ABI®), the AmpliSensor™ (InGen), and the Sunrise™ (Oncor®, Appligene®) systems.
The system in most widespread use is the TaqMan™ system.
That procedure combines activities of DNA polymerase and the 5′→3′ nuclease of Taq polymerase during PCR. The principle is as follows: in addition to the two primers with a sequence complementary to that of the target to be amplified, a probe, the reporter probe, is added to the reaction medium. It has the ability to hybridise with the target in the body of the amplified sequence, but cannot itself be amplified. A phosphoryl group added to the 3′ end of the probe prevents it from being extended by Taq polymerase. A fluorescein derivative and a rhodamine derivative are incorporated into the-probe, respectively at the 5′ and 3′ ends. The probe is small, so the rhodamine derivative located close to the fluorescein absorbs the energy emitted by the fluorescein when it is excited (quenching).
Once the primers are hybridised to the target, during the elongation reaction, Taq DNA polymerase attacks the probe via its 5′ nuclease activity, releasing the quencher group and thus re-establishing fluorescence. The intensity of the emitted fluorescence is then proportional to the quantity of PCR products formed, which provides a quantitative result. The emitted fluorescence is proportional to the initial number of target molecules. The fluorescence development kinetics can be followed in real-time during the amplification reaction.
That technique has the advantage of being capable of ready automation. An instrument that can carry out the technique, the ABI Prism 7700™, is sold by Perkin-Elmer. That instrument combines a thermocycler and a fluorimeter. It can detect the increase in fluorescence generated during a quantification test using the TaqMan™ procedure, by means of optical fibres located under each tube and connected to a CCD camera that detects, in real-time, the signal emitted by the fluorescent groups liberated during PCR. Quantitative data are deduced by determining the cycle at which the signal from the amplification product reaches a certain threshold determined by the operator. Several studies have demonstrated that the number of cycles is proportional to the quantity of initial material (Gibson, Heid et al., 1996; Heid, Stevens et al., 1996; Williams, Giles et al., 1998).
The number of potential applications of such an instrument is considerable, in human health, in the agroalimentary field and in quality control. Unfortunately, the ABI Prism 7700™ and the several other competing instruments currently on the market are extremely expensive. Further, they can only be used by a trained operator. In practice, such instruments are only used in certain highly specialised areas.
Thus, there is a need for a nucleic acid amplification system, if necessary measuring in real-time, which does not have the disadvantages of the prior art mentioned above.